The association and dissociation of proteins is crucial to all aspects of cell function. Examples of protein-protein interactions are evident in hormones and their respective receptors, in intracellular and extracellular signaling events mediated by proteins, in enzyme substrate interactions, in intracellular protein trafficking, in the formation of complex structures like ribosomes, viral coat proteins, and filaments, and in antigen-antibody interactions. Intracellular assays for detection of protein interactions and identification of their inhibitors have received wide attention with the completion of the human genome sequence.
One method of identifying protein-protein interactions is the yeast two-hybrid system (Fields and Song, 1989, Nature, 340:245-246). This assay utilizes the reconstitution of a transcriptional activator like GAL4 (Johnston, 1987, Microbiol. Rev., 51:458-476) through the interaction of two protein domains that have been fused to the two functional units of the transcriptional activator: the DNA-binding domain and the activation domain. This is possible due to the bipartite nature of certain transcription factors like GAL4. Being characterized-as bipartite signifies that the DNA-binding and activation functions reside in separate domains and can function in trans (Keegan et al., 1986. Science 231:699-704). The reconstitution of the transcriptional activator is monitored by the activation of a reporter gene like the lacZ gene that is under the influence of a promoter that contains a binding site for the DNA-binding domain of the transcriptional activator. This method is most commonly used either to detect an interaction between two known proteins (Fields and Song, 1989, Nature, 340:245-246) or to identify interacting proteins from a population that would bind to a known protein (Durfee et al., 1993, Genes Dev., 7:555-569; Gyuris et al., 1993, Cell, 75:791-803; Harper et al, 1993, Cell, 75:805-816; Vojtek et al., 1993, Cell, 74:205-214).
In the yeast two-hybrid system, the protein interaction must occur in yeast and must occur in the nucleus of the yeast. In addition, the yeast two hybrid assay suffers from a long lag time between assay start and detection of results, false positives and lack of control over the conditions wherein the binding partners interact. Furthermore, this system is limited to detecting protein-protein interactions. What are needed are simple, robust methods for detecting molecular interactions in a variety of situations. In particular, what is needed is an in vitro method that further amplifies the interaction signal.